Use of c1 inhibitor for the prevention of ischemia-reperfusion injury

ABSTRACT

The present invention relates to the therapeutic and prophylactic use of C1 inhibitor for preventing, reducing and treating ischemia and reperfusion injury. The C1 inhibitor of the present invention is still therapeutically effective when administered after an ischemic period and reperfusion and therefore particularly useful for unforeseen occurrences of ischemic reperfusion such as e.g. a stroke.

FIELD OF THE INVENTION

The present invention relates to the therapeutic and prophylactic use ofC1 inhibitor for preventing, reducing and treating ischemia-reperfusioninjury, particularly cerebral ischemia-reperfusion injury that may occuras a result of a stroke.

BACKGROUND OF THE INVENTION

Ischemia-reperfusion injury is a well known occurring pathologiccondition. It may either represent a foreseen pathologic condition or anunforeseen pathologic condition. Stroke is one of the most common typesof unforeseen ischemia-reperfusion injury. Stroke is the third cause ofdeath and the leading cause of long-term disability in industrializedcountries. Stroke is a type of cardiovascular disease that affects thearteries leading to and within the brain. A stroke occurs when sucharteries are blocked by a clot or bursts and results in ischemia of thecerebral tissues that are served by the blocked artery. Direct damage tothe brain is caused by the interruption of the blood flow, mainly due toloss of oxygenation to the viable tissue, ultimately leading toinfarction if not reversed. However if the insult is reversed (eitherphysiologically or therapeutically) then reperfusion of the ischemictissue may paradoxically cause further indirect damage. When there is along duration of ischemia, the “direct” damage resulting from hypoxiaalone is the predominant mechanism. For shorter duration's of ischemia,the indirect or reperfusion mediated damage becomes increasingly moreimportant to the final outcome.

C1 inhibitor (C1INH), the inhibitor of complement C1, has been reportedto display neuro-protective action by reducing ischemia-reperfusioninjury in rodent models for cerebral ischemia-reperfusion. (De Simoni etal., 2003, J Cereb Blood Flow Metab. 23: 232-9; Akita et al., 2003,Neurosurgery 52: 395-400). The neuro-protective action of C1INH on brainischemia-reperfusion injury does not require C1q (De Simoni et al.,2004, Am J Pathol. 164: 1857-63). More recently Storini et al. (2005,Neurobiol Dis. 19: 10-7) reported that C1INH exerts an anti-inflammatoryand anti-apoptotic action on ischemia-reperfusion injury throughinhibition of inflammation and cell recruitment from the vasculature tothe ischemic site. However, the window in time around the stroke duringwhich administration of C1INH is therapeutically effective is rathernarrow. It is therefore an object of the present invention to providefor C1INH with a broader window in terms of time of administration.

DESCRIPTION OF THE INVENTION

The present invention is based on the surprising finding that wherenaturally occurring plasma derived C1 inhibitor (C1INH), has lost mostof its ability to reduce ischemia reperfusion injury in a mouse modelfor transient cerebral focal ischemia when administered after ischemia,a recombinant preparation of C1INH is still able to exert itsneuroprotective effects also when injected at least 1 hour afterischemia and/or reperfusion. Surprisingly, a neuroprotective effect canstill be reached when the C1INH is injected 18 hours after ischemiaand/or reperfusion. The difference between the naturally occurringplasma derived C1INH and the recombinant preparation of C1INH is thatthe first has a plasma half life of at least 24 hours and is fullysialylated glycoprotein, and the latter has a reduced plasma half lifeand has a different glycosylation as compared to the plasma derivedproduct.

A difference known between the naturally occurring plasma derived C1INHand the recombinant preparation of C1INH is the extent and type ofglycosylation. The recombinant glycoprotein contains a broad array ofdifferent N-glycans, comprising oligomannose-, hybrid-, and complex-typestructures, whereas the N-glycans of plasma derived C1INH are mainlycomposed of fully sialylated complex-type structures. As a result of thedifferences in glycosylation, the plasma derived glycoprotein has aplasma half life of at least 24 hours and the recombinant C1INH has areduced plasma half life.

In one aspect the present invention therefore relates to a method forthe prevention, reduction or treatment of at least one of ischemia andreperfusion injury, whereby the C1 inhibitor is administered after theischemia and/or after the reperfusion. The method preferably comprisesthe step of administering an effective amount of a C1INH having a plasmahalf life that less than the plasma half life of a plasma derived C1INH.Alternatively, the method preferably comprises the step of administeringan effective amount of a C1INH that has a different glycosylation ascompared to the plasma derived C1INH. This method relates to atherapeutic and/or prophylactic use of C1 inhibitor for preventing,reducing and/or treating any type of ischemia-reperfusion injury.

A C1 inhibitor, also referred to as C1 esterase inhibitor is hereindefined, as an inhibitor of complement C1. C1INH belongs to thesuperfamily of serine proteinase inhibitors and is the only inhibitor ofC1r and C1s of the complement system and is the major inhibitor offactor XIIa and kallikrein of the contact system. In addition C1INH alsoinhibits other serine proteases of the coagulation and fibrinolyticsystems like factor XI, tissue type plasminogen activator and plasmin(Schapira et al. 1985, Complement 2: 111; Davis, 1988, Ann Rev. Immunol.6: 595). Human C1INH is a protein of 500 amino acids, including a 22amino acid signal sequence (Carter et al. 1988, Euro. J. Biochem. 173;163). Plasma C1INH is a glycoprotein of approximately 76 kDa and isheavily glycosylated, up to 26% of its molecular mass consists ofcarbohydrate (Perkins et al., 1990, J. Mol. Biol. 214, 751). A C1INH foruse in the methods of the present invention preferably is a protein withan amino acid sequence that has at least 65, 67, 68, 69, 70, 75, 80, 85,90, 95, 98 or 99% identity with the amino acid sequence of the maturehuman C1INH as depicted in SEQ ID NO:1.

For the purpose of the present invention, the degree of identity betweentwo amino acid sequences refers to the percentage of amino acids thatare identical between the two sequences. First, homologous polypeptidesequences are searched using the Basic Local Alignment Search Tool(BLAST) algorithm, which is described in Altschul, et al., J. Mol. Biol.215: 403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The BLAST algorithm parameters W, B, andE determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 3, the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4. Next,the degree of identity (as defined above) of homologous sequences isdetermined using the CLUSTALW alignment algorithm (Higgins D. et al(1994). Nucleic Acids Res. 22:4673-4680) using the following parameters;Gap size: 5, Gap open: 11, Gap extension: 1, Mismatch: −15, Word size:3.

The C1INH preferably has C1INH activity as may e.g. be assayed asdescribed by Drouet et al. (1988, Clin Chim Acta. 174:121-30). Morepreferably, the C1INH is a human C1INH (hC1INH) which is understood tomean that the C1INH has an amino acid sequence that naturally occurs inman (as e.g. SEQ ID NO:1 or CAA30314) but does not mean that the C1INHis produced in and obtained from e.g. human plasma.

According to one aspect of the invention the C1INH for use in themethods of the invention preferably has a reduced plasma half life ascompared to the plasma half life of plasma derived C1INH, morepreferably the plasma half life of the C1INH of the invention is lessthan the plasma half life of C1INH derived from human plasma. By “areduced plasma half life” is meant the negative change in circulatinghalf life of a C1INH of the invention relative to the circulating halflife of a plasma derived C1INH. In this context, a plasma derived C1INHrefers to naturally occurring C1INH which is typically derived fromplasma and which may be purified from plasma but is not modified inchemically or enzymatically.

Plasma half life is measured by taking blood samples at various timepoints after administration of the C1INH, and determining theconcentration of the C1INH in each sample. Correlation of the serumconcentration with time allows calculation of the plasma half life. Thereduction of plasma half life of a C1INH of the invention relative tothe circulating half life of a plasma derived C1INH preferably is atleast about two-fold, at least about three-fold, at least aboutfour-fold, at least about six-fold, more preferably at least abouteight-fold, and most preferably at least about ten-fold. In other words,plasma half life of a C1INH of the invention preferably is less than 60,50, 40, 30, 25, 20, 15, 12.5 or 10% of the plasma half life of a plasmaderived C1INH, i.e. its naturally occurring counterpart.

E.g. the plasma half life of the C1INH of the invention that is used inthe Examples herein, which is obtained from the milk of transgenicrabbits, exhibits a plasma half life in humans of about 3 hours, whichabout four- to eight-fold less than the average plasma half life of aplasma derived C1INH in man. It is understood that the determination ofthe reduction of plasma half life of a C1INH of the invention ascompared to that of plasma derived C1INH is preferably performed undersimilar if not identical conditions, i.e. preferably at correspondingdosages, sampling regimes, in the same organism, which may be alaboratory animal such as a mouse or human subjects, and in about thesame number of test subjects. Furthermore, it is understood that theaverage plasma half lives of both C1INH preparation are compared as maybe determined by standard method of statistical analysis.

A C1INH with shorter half life, be it a naturally occurring or arecombinantly produced C1INH, may be prepared by any convenient method.It may for example be prepared in vivo in a recombinant host cell ororganism that results in a C1INH with a modified carbohydrate structure(as compared to the plasma derived C1INH) or the carbohydrate structureof a naturally occurring C1INH may be chemically or enzymaticallymodified in vitro. Preferably, the C1INH of the invention is modifiedcompared to the plasma derived C1INH the following way: removal of acarbohydrate moiety (from a naturally occurring variant or recombinantlyexpressed variant of the glycoprotein), preferably the removal of sialicacid and/or galactose from a N-linked carbohydrate chain and/or theremoval of a carbohydrate chain resulting in exposure of mannose,galactose, N-acetylglucosamine and/or fucose residues.

According to another aspect of the invention the C1INH for use in themethods of the invention preferably has a different glycosylation ascompared to the plasma derived C1INH. Modifications to the carbohydratestructure of a C1INH of the invention include modifications which leadto underglycosylation, overglycosylation, to the asialio form of C1INH,or any other modifications which lead to a different glycosylationpattern.

In vitro, underglycosylation may be the result of a deletion of acarbohydrate moiety or of a complete carbohydrate chain of C1INH.Modifications may involve both N- or O-linked carbohydrate chains, oronly one type of chain. It may involve all the chains, or only some ofthe chains. Overglycosylation may for instance be the result of theaddition of an extra carbohydrate moiety or a complete carbohydratechain to the C1INH molecule. An asialo-form of C1INH or a form having areduced level of terminal sialic acid residues may typically be obtainedby removal of a sialic acid group. It is well-known that the half lifeof a glycoprotein in the blood is highly dependent on the compositionand structure of its N- and O-linked carbohydrate groups. In general,maximal half life of a glycoprotein requires that its N- and O-linkedcarbohydrate groups have a terminal sialic acid. If this terminal sialicacid is not present, the glycoprotein is rapidly cleared from the blooddue to the exposure of galactose residues. It is well-established thatthe presence of terminal galactose residues in carbohydrate moieties ofglycoproteins results in enhanced plasma clearance by theasialoglycoprotein receptor in the liver. Thus in a preferredembodiment, C1INH for use in the methods of the present inventionpreferably has a reduced level of terminal sialic acid residues ascompared to plasma derived human C1 inhibitor. Sialic acid may beremoved in several ways. For instance, it may be removed chemically orenzymatically, for example, by treatment with sialidase. Suitablesialidases for this purpose are described by Chou et al. (1996, J BiolChem. 271(32):19219-24; and 1994, J Biol Chem. 269(29):18821-6) and maye.g. be obtained from V-labs, Inc. (Covington, La., USA). In a furtherpreferred embodiment, C1INH for use in the methods of the presentinvention preferably has exposed mannose, N-acetylglucosaminephosphomannose, galactose and/or N-acetylgalactosamine residues. Anexposed sugar residue will usually be a terminal sugar residue on aglycan branch or at least a sugar residue that is accessible forinteractions with a moiety having affinity for the residue (such as acarbohydrate binding domain). A C1INH with exposed galactose,N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose orphosphomannose residues may e.g. be obtained by enzymatic treatment withone or more of β-D-N-acetylhexosaminidase, endo-β-D-galactosidase,and/or α-D-N-acetylgalactosaminidase (also obtainable form e.g. V-labs,Inc., Covington, La., USA).

In vivo, modifications of carbohydrate chains of C1INH may be introducedby using recombinant production systems. Both prokaryotic and eukaryoticcell cultures may be used, such as yeast cells, fungal cells, insectcells and mammalian cells. For example, COS cells and CHO cells aresuitable mammalian production systems. Although mammalian cell culturesystems have the capacity to produce glycoproteins with sialylatedcarbohydrate groups, optimal, natural or complete glycosylation is oftendifficult to achieve and consequently, recombinantly producedglycoproteins in general have a different glycosylation pattern thantheir natural counterparts. Usually this different glycosylation patternis incomplete (as compared to the natural counterparts) having exposedgalactose, N-acetylglucosamine and/or mannose residues. Likewise,production of C1INH in eukaryotic microorganisms like yeasts or fungiwill result in C1INH with exposed mannose residues.

C1INH with modified carbohydrate structures may also be prepared intransgenic animals, preferably in non-human animals, such as intransgenic rabbits, bovine, mice, rats, goats and sheep. Preferably,such glycoproteins are expressed in the mammary glands of thesenon-human transgenic animals such that the glycoproteins may be obtainedfrom the milk of the animal. The skilled person will understand that itwill depend on the specific glycoprotein to be produced and on theamount which has to be produced, which transgenic animal is best usedfor production. A particularly preferred C1INH for use in the presentinvention is a C1INH that is obtained from the milk of a transgenicbovine or an animal of the order Lagomorpha, preferably of the familyLeporidae, more preferably of the genus Oryctolagus and most preferablya rabbit of the species Oryctolagus cuniculus.

Different types of modifications to the structure of the carbohydratechain of the C1INH protein as compared to its natural plasma-derivedcounterpart may be obtained from recombinant production systems, such asdifferent glycosylation, underglycosylation or overglycosylation may beintroduced separately or in combination, simultaneously orconsecutively, some types may be introduced to one part of the molecule,while others are introduced to another part of the molecule. Preferredcombinations of modifications contribute to the therapeutic efficacy ofthe protein include exposed galactose, N-acetylgalactosamine,N-acetylglucosamine, mannose, fucose and/or phosphomannose residues onthe C1INH of the invention. The C1INH of the invention may e.g. haveglycans of the oligomannose type or of the highmannose type. Preferablyat least about 5, 10, 15, 20, 40 or 60% of the terminal residues on theglycans on the C1INH are selected from galactose, N-acetylgalactosamine,N-acetylglucoseamine, mannose, fucose and phosphomannose residues. E.g.a preferred C1INH for use in the present invention contains about 2, 4,5, 6-fold less sialic acid as compared to its natural counterpart and/orat least about 5, 10, 15, 20, 40 or 60% of its N-linked glycans areneutral carrying terminal hexoses with equatorial 3- and 4-OH groups,such as mannose and N-acetylglucosamine. In contrast, plasma derivedC1INH has no oligomannose type glycosylation. A preferred C1INH for usein the present invention e.g. is a recombinant human C1INH produced inthe mammary glands of rabbits which has 5-6 fold less sialic acid ascompared to its natural counterpart and about 15% of its N-linkedglycans are neutral carrying terminal mannose residues.

In a preferred embodiment, the different glycosylation of the C1INH foruse in the present invention results in a higher affinity for a mannosebinding protein as compared to its plasma derived counterpart. Themannose binding protein (MBP) is also referred to as mannan-bindingprotein, mannose-binding lectin (MBL), mannan-binding lectin, orbactericidal Ra-reactive factor. MBP is a collectin that belongs to agroup of soluble Ca²⁺-dependent (C-type) lectins. MBP is an activator ofcomplement via the lectin pathway (that differs from the classical andalternative pathways of complement activation). The complement system isan important component of the innate immune defense and is activated bythree pathways: the classical pathway, the alternative pathway, and therecently discovered lectin or Mannose binding lectin (MBL) pathway.

The activation of the classical pathway begins when the catalyticdomains C1r and C1s bind to immune complexes via the recognition proteinC1q (see FIG. 11).

The alternative pathway is continuously turning over at a slow rate inan antibody-independent manner and will attack particles that are notspecifically protected against complement.

The lectin or MBL pathway is initiated or activated upon binding of MBLto carbohydrate structures present on various pathogens or othercellular structures. Two serine proteases: mannan-binding lectinassociated serine protease (MASP)-1 and -2 (see FIG. 11) are associatedwith MBL and show striking similarities with the serine proteases C1sand C1r. The complex has C4- and C3-activating capacities upon bindingto mannan. The complex contains two serine proteases MASP-1 and MASP-2linked by a disulfide bond. In this form, MASP is capable of cleaving C4and C3 resulting in their activation. The C1INH of the inventionpreferably has a higher affinity for a human MBP as compared to itsplasma derived counterpart.

MBP recognizes exposed hexoses with equatorial 3- and 4-OH groups, suchas mannose and N-acetylglucosamine and/or N-acetyl-hexosamines. Apreferred C1INH of the invention therefore carries such terminalhexoses. The higher affinity for MBP, preferably human MBP of the C1INHof the invention preferably is such that it allows a more efficienttargeting, binding and/or inhibition of MBP as compared to its naturalplasma derived counterpart that lacks exposed mannose andN-acetylglucosamine residues. Human MBP is herein understood to refer tothe protein characterized by Kawasaki et al. (1983, J. Biochem94:937-47), having an amino acid sequence as described by Taylor et al.(1989, Biochem. J. 262 (3), 763-771; NCBI accession no. CAA34079). Thestructure of rat MBP complexed with an oligosaccharide is described byWeis et al. (1992, Nature. 360:127-34). For a further description ofhuman MBP see e.g U.S. Pat. No. 6,846,649 and references cited therein.

All of these pathways (classical, alternative and lectin or MBL)generate a crucial enzymatic activity that eventually leads to theassembly of the membrane attack complex (MAC or C5b-C9) (see FIG. 11).Under physiological conditions, activation of the complement system iseffectively controlled by the coordinated action of soluble andmembrane-associated regulatory proteins. One of these proteins is C1inhibitor (C1INH), a serine protease inhibitor that binds to C1s and C1rand currently the only known physiological inhibitor of the classicalpathway. In addition, C1INH is able to inactivate MBL-mediatedcomplement activation by binding and inhibiting MASP-1 and MASP-2.

The activation of the different complement pathways is preferablymeasured in human sera by the Wielisa kit (product no. COMPL 300,Wieslab, Sweeden). This is a commercially available enzyme immuno assay,specific for the detection of each of the three complement pathways withdeposition of C5b-C9 as a common read-out. Briefly, wells of microtitrestrips are coated with specific activators of each of the threecomplement pathways. Human serum is diluted in diluent containingspecific blocker to ensure that only the respective pathway isactivated. C1INH of the invention or its plasma-derived counterpart isfurther added in a concentration ranged between 0 and 75 μmol, incubatedfor 30 minutes at room temperature and added to the wells. During asubsequent incubation of the diluted human serum in the well for 60minutes at 37° C., complement is activated by the specific coating. Thewells are then washed and C5b-C9 formed is detected with a specificalkaline phosphatase labelled anti C5b-C9 antibody. After a furtherwashing step, detection of specific antibodies is obtained by incubationwith alkaline phosphatase substrate solution. The amount of complementactivation correlates with the colour intensity and is measured in termsof absorbance (optical density OD). Using this kit, both recombinanthuman C1INH (rhC1INH) of the invention and plasma-derived C1INH(pdC1INH) were found to have similar inhibiting capacities for theclassical pathway. However, the C1INH of the invention was found to haveapproximately 20% more inhibiting capacity for the MBL pathway thanplasma-derived C1INH (see example 3).

Therefore accordingly, in this preferred embodiment, the differentglycosylation of the C1INH for use in the present invention results in ahigher affinity for a MBP as compared to its plasma derived counterpart,which results in a more efficient inhibition of MBP, leading to a moreefficient inhibition of the lectin pathway. More efficient inhibition ofthe lectin pathway preferably means at least 5% more inhibition, evenmore preferably at least 10% more inhibition, even more preferably atleast 15% more inhibition even more preferably at least 20% even morepreferably at least 25% even more preferably at least 30% even morepreferably at least 35% even more preferably at least 40% even morepreferably at least 45% even more preferably at least 50% even morepreferably at least 55% even more preferably at least 60% even morepreferably at least 65% even more preferably at least 70% even morepreferably at least 75% even more preferably at least 80% even morepreferably at least 85% even more preferably at least 90% even morepreferably at least 95% and most preferably at least 98% moreinhibition. The activation of the lectin pathway is preferably measuredby the Wielisa kit as described above.

The method of the invention may be applied to prevent, reduce or treatany type of ischemia and reperfusion injury. Preferably, the method ofthe invention is applied wherein the ischemia and reperfusion injury isknown to arise at least in part, more preferably mostly via the lectinpathway. For myocardial ischemia and reperfusion injury (J Immunology2005, 175: 541-546), renal ischemia-reperfusion injury (Am J Pathol.2004 165(5):1677-88), gastrointestinal ischemia reperfusion injury (JImmunol. 2005 15:174(10):6373-80), and for stroke (deSimoni et al, 2004Am J. Pathol. 164:1857-63) it has been shown that reperfusion injuryarises mostly via the lectin pathway and hardly via the classicalpathway. Hence, a C1INH of the invention preferably is a more potentinhibitor of the lectin pathway as compared to its natural plasmaderived counterpart. Preferably a C1INH of the invention is a morepotent in vivo inhibitor of the lectin pathway in man as compared to itsnatural plasma derived counterpart.

Unlike the experimental model used in the Examples herein, theoccurrence of ischemia in real life often is an unforeseen event.Therefore administration of C1INH prior to the occurrence of ischemiaand/or subsequent reperfusion is not generally a feasible option andinevitably in practice C1INH will have to be administered some time ifnot several hours after ischemia and/or subsequent reperfusion. This,however, seriously limits the therapeutic usefulness of conventionalplasma derived C1INH because it is mostly ineffective when administeredsubsequent to ischemic reperfusion and only has a very small time windowfor therapeutic efficacy (see FIG. 2 and deSimoni et al, 2004 Am J.Pathol. 164:1857-63). In contrast, a C1INH for use in the presentinvention as defined above, is still able to exert its neuroprotectiveeffects also when injected at least 1 hour after ischemia or after theonset of ischemia and/or 30 minutes after the start of the reperfusion.Therefore, in a preferred embodiment of the method for the prevention,reduction or treatment of at least one of unforeseen or foreseenoccurrence of ischemia and reperfusion injury, the C1INH of theinvention is administered at least at the end or after the ischemicperiod, i.e. when the ischemic tissue is reperfused. More preferably,the C1INH of the invention is administered at least 10, 15, 20, 30, 45,60, 90 or 120 minutes after the ischemic period or after the start ofreperfusion. Preferably, the C1INH of the invention is administered nomore than 24, 12, 6, 4 or 3 hours after ischemia or after the onset ofischemia and/or reperfusion. In another preferred embodiment, the C1inhibitor is administered at least 3 hours after ischemia or after theonset of ischemia and/or reperfusion, preferably at least 6 hours, morepreferably at least 9 hours, even more preferably at least 18 hours.

In one preferred embodiment, the method is applied to prevent, reduce ortreat an unforeseen, sudden or acute occurrence of ischemic reperfusion.Conditions and disorders associated with an unforeseen, sudden or acuteoccurrence of ischemic reperfusion injury include but are not limited toischemic reperfusion injury after acute myocardial infarction (AMI),after stroke, including perinatal stroke, after hemorrhagic shock, afterintestinal ischemia, after emergency coronary surgery for failedpercutaneous transluminal coronary angioplasty (PCTA), after anyvascular surgery with blood vessel cross clamping (e.g. of aorta,leading to skeletal muscle ischemia), or after pancreatitis aftermanipulation of pancreatic or bile duct (ERCP). In such instances theC1INH of the invention preferably is administered at least 1, 5, 10, 15,20, 30, 45, 60, 90 or 120 minutes after the acute myocardial infarction(AMI), after stroke, including perinatal stroke, after hemorrhagicshock, after intestinal ischemia, after emergency coronary surgery forfailed percutaneous transluminal coronary angioplasty (PCTA), after anyvascular surgery with blood vessel cross clamping (e.g. of aorta,leading to skeletal muscle ischemia), or after pancreatitis aftermanipulation of pancreatic or bile duct (ERCP). Alternatively, the timeof administering the C1INH of the invention may be defined as preferablyat least 1, 5, 10, 15, 20, 30, 45, 60, 90 or 120 minutes after the startof reperfusion.

In addition, unforeseen ischemic reperfusion injury is preferablydefined as an ischemic reperfusion injury wherein a therapy or surgeryinduces a reperfusion but not an ischemia. Such therapy or surgeryinclude but not limited to:

-   -   pharmacological thrombolysis, including intravenous and        endovascular therapies for stroke, acute coronary syndromes,        peripheral arterial occlusion, pulmonary embolus, renal artery        occlusion,    -   mechanical thrombolysis, e.g. percutaneous coronary        intervention, peripheral arterial angioplasty, visceral arterial        angioplasty,    -   coronary artery bypass grafting,    -   carotid endarterectomy,    -   mesenteric ischemia,    -   shock including hemorrhagic, cardiogenic, neurogenic,        analphylactic,    -   flap-failure, e.g. plastic surgery,    -   re-implantation of digits and limbs,    -   strangulated bowel.

Alternatively, in another preferred embodiment, the method is applied toprevent, reduce or treat a foreseen occurrence of ischemic reperfusion.A foreseen occurrence of ischemia reperfusion injury preferably includesa setting in which a therapy or surgery induce both an ischemia andsubsequently a reperfusion. A non-limiting list is given below oftherapy or surgery in which there is an induced temporary period of noor low blood flow, i.e. ischemia or hypoxia, followed by reperfusion:

-   -   cardiopulmonary bypass,    -   aneurysm repair, including aortic, cerebral,    -   carotid endarterectomy in which a clamp is used during the        surgery,    -   deep hypothermic circulatory arrest,    -   tourniquet use, i.e. in trauma settings,    -   solid organ transplantation,    -   any other iatrogenic disruption of blood flow.

In addition, conditions and disorders associated with a foreseenoccurrence of ischemic reperfusion injury include but are not limited toischemic reperfusion injury after organ transplantation (lung, liver,kidney, heart), after any vascular surgery with blood vessel crossclamping (e.g. of aorta, leading to skeletal muscle ischemia), or afterpancreatitis after manipulation of pancreatic or bile duct (ERCP), afteror during extra corporal circulation (ECC).

In a preferred embodiment of the method for the prevention, reduction ortreatment of at least one of foreseen occurrence of ischemia andreperfusion injury, the C1INH of the invention is administered at leastat the end or after the ischemic period, i.e. when the ischemic tissueis reperfused. More preferably, the C1INH of the invention isadministered at least 10, 15, 20, 30, 45, 60, 90 or 120 minutes afterthe ischemic period or after the start of reperfusion. Preferably, theC1INH of the invention is administered no more than 24, 12, 6, 4 or 3hours after ischemia or after the onset of ischemia and/or reperfusion.In another preferred embodiment, the C1 inhibitor is administered atleast one hour after ischemia or after the onset of inschemia and/orreperfusion, 3 hours after ischemia or after the onset of ischemiaand/or reperfusion, preferably at least 6 hours, more preferably atleast 9 hours, even more preferably at least 18 hours.

Alternatively, in another aspect of the invention, a method is providedfor the prevention, reduction or treatment of at least one of foreseenoccurrence of ischemia and reperfusion injury, wherein the C1 INH of theinvention is administered before or during the ischemia and reperfusion.The skilled person will understand that depending upon the plasma halflife of the C1INH of the invention, the earliest possible time point,wherein the C1 INH of the invention may be administered may be adjustedto obtain the best possible result.

According to one preferred embodiment, the C1INH of the invention iscontinuously administered to a subject in the need thereof and/or incase of an organ transplantation to the organ to be transplanted. Theorgan to be transplanted is preferably conserved in a composition with asuitable medium and suitable amount of C1INH.

Alternatively or in combination with former preferred embodiment, beforethe occurrence of a foreseen type of ischemia and reperfusion injurypreferably means that the administration is performed at the most 3hours before at least one foreseen occurrence of ischemia andreperfusion injury, preferably at the most 2 hours, more preferably atthe most one hour, and most preferably at the most 30 minutes.

A subject in the need thereof is a subject wherein a foreseen occurrenceof ischemia and reperfusion injuries may occur. Foreseen occurrence ofischemia and reperfusion injuries have been already described herein.

The administration of the C1INH before the foreseen occurrence ofischemia and reperfusion injury is attractive since it may prevent theoccurrence of most if not all damages associated with the ischemia andreperfusion injury the same way as presented when the C1INH isadministered after the occurrence of ischemia and reperfusion injury, ifnot better.

In a more preferred embodiment, the method is applied to an unforeseenoccurrence of ischemic reperfusion. Even more preferably, an ischemicreperfusion injury occurring after a stroke or a perinatal stroke. Inthese types of unforeseen occurrence of ischemic reperfusion, wedemonstrated that the C1 inhibitor of the invention exerts aneuroprotective effect in the ischemic penumbra. The ischemic penumbrapreferably means the hippocampus and/or cortex. A neuroprotective effectpreferably means that neurodegeneration is counteracted in thehippocampus and/or cortex after treatment with the C1 inhibitor of theinvention up to 3 hours after the onset of ischemia in the hippocampusand up to 9 hours after the onset of ischemia in the cortex. Morepreferably, neurodegeneration is counteracted up to 4, 5, 6 hours ormore in the hippocampus and up to 10, 11, 12 hours or more in theischemia. Neurodegeneration is preferably assessed as in example 2:brain sections are stained with a marker specific for neuronaldegeneration, preferably Jade (Schmued L C, et al, reference 4) andanalyzed by fluorescent microscopy. Using this method, counteraction ofneurodegeneration means at least 2% less stained cells are visualized inthe treated sample compared to the untreated sample. Preferably,counteraction of neurodegeneration means at least 5% less stained cells,at least 7%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40% or more.

Alternatively or in combination with former mentioned embodiment, theuse of the C1 inhibitor exerts a reduction of the lesion induced by theischemia and/or reperfusion. More preferably, when the ischemicreperfusion injury occurred after a stroke or a perinatal stroke, theuse of the C1 inhibitor of the invention exerts a reduction of theinfarct size. Even more preferably, the infarct size is quantified aspresented in example 2. Even more preferably, using this quantificationmethod, at least 3 hours after the onset of ischemia, a reduction of atleast 10% of the infarct size is reached, even more preferably at least20%, even more preferably at least 40%, even more preferably at least60%, even more preferably at least 70%, even more preferably at least80%, and most preferably at least 90%.

A C1INH for use in the methods of the invention may be part of orcombined with state of the art pharmaceutical compositions. Thesepharmaceutical compositions typically comprise the C1INH and apharmaceutically acceptable carrier or excipient. These pharmaceuticalcompositions may be administered in a number of ways depending onwhether local or systemic treatment is desired, the area to be treatedand the stability of the active compound. Suitable formulations willdepend on the method of administration. The pharmaceutical compositionis preferably administered by par-enteral administration, such as forexample by intravenous, intra-arterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or by intrathecal or intracranialadministration. In a preferred embodiment it is administered byintravenous infusion. Suitable formulations for parenteraladministration are known in the art and are typically liquidformulations. C1INH preparations for parental administration must besterile. Sterilization is readily accomplished by filtration throughsterile filtration membranes, prior to or following lyophilization andreconstitution. C1INH preparations may be administered continuously byinfusion or by bolus injection. Liquid C1INH formulations may forexample be administered by an infusion pump. A typical composition forintravenous infusion could be made up to contain 100 to 500 ml ofsterile 0.9% NaCl or 5% glucose optionally supplemented with a 20%albumin solution and 100 to 500 mg of the C1INH. A typicalpharmaceutical composition for intramuscular injection would be made upto contain, for example, 1-10 ml of sterile buffered water and 1 to 250mg of the C1INH of the present invention. Methods for preparingparenterally administrable compositions are well known in the art anddescribed in more detail in various sources, including, for example,Remington's Pharmaceutical Science (15th ed., Mack Publishing, Easton,Pa., 1980) (incorporated by reference in its entirety for all purposes).

The effective dose, i.e. effective concentration and frequency, of theC1INH when used in the methods of the invention will depend on thespecific pharmaceutical composition which is used, the severity of thecondition and the general state of the patient's health. In general, theeffective dose of a pharmaceutical composition which is based on a C1INHfor use in the methods of the invention may be found by routineoptimisation. A suitable starting point is the dose which is used forthe equivalent pharmaceutical composition which is based onplasma-derived C1INH. A great advantage of a pharmaceutical compositionof the invention is that a high initial dose may be used in treatment,which enhances the likelihood of successful treatment. This high initialdose is possible because the C1INH in the pharmaceutical composition ofthe invention shows a faster clearance than its natural counterpart. Inparticular for the treatment of acute cases, a high initial dose of theC1INH of the invention may be advantageous. This high initial dose maybe at least 1.5, at least 2, 3 or 4 times the dose of the naturaloccurring counterpart which would be administered.

In a preferred embodiment, C1INH of the invention is administeredintravenously at a dose of more than 50, 100, 200, 400, 600, 800, or1000 U/kg body weight of the individual, preferably in the range of50-2000, 100-1000, 200-800, 400-700 or 500-700 U/kg body weight of theindividual. One unit (U) of C1INH is the amount of C1INH present in 1millilitre of human blood. One such unit corresponds to approximately275 microgram plasma derived C1INH. Assuming a molecular weight of110,000 dalton, the concentration in human plasma of C1INH is 2.5micromol per litre (Nuijens et al. (1989), J. Clin. Invest. 84:443).

In a further preferred embodiment of the method of the invention thepharmaceutical composition further contains a thrombolytic agent or isfor use in combination with a thrombolytic agent or after subsequenttreatment with such agent. A thrombolytic agent is herein understood tomean an agent (drug) that is able to dissolve a blood clot (thrombus)and reopen an artery or vein. Thrombolytic agents are usually serineproteases and convert plasminogen to plasmin which breaks down thefibrinogen and fibrin and dissolves the clot. Preferred thrombolyicagents include reteplase (r-PA or Retavase), alteplase (t-PA orActivase), urokinase (Abbokinase), prourokinase, anisoylated purifiedstreptokinase activator complex (APSAC), and streptokinase.

In a further aspect, particularly for jurisdictions other than the USA,the invention pertains to the use of a C1INH of the invention as definedherein above for the manufacture of a medicament for the prevention,reduction or treatment of reperfusion injury in accordance with any ofthe methods defined herein above.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

DESCRIPTION OF THE FIGURES

FIG. 1 Assessment of infarct size forty-eight hours after ischemia inmice treated with saline or with 15 U rhC1INH (recombinant human C1INH,see Example 1.2) per mouse pre, post and 1 h post ischemia.

FIG. 2 Assessment of infarct size twenty-four hours after ischemia inmice treated with saline or with 15 U plasma derived hC1INH per mousepre, post and 1 h post ischemia.

FIG. 3 Infarct volume assessed 48 h after ischemia in mice receivingsaline or 15 U/mouse of rabbit rhC1-INH at different time points fromthe beginning of ischemia. Data are expressed as mean±SEM (n=6 mice pergroup). *P<0.05, **P<0.01 versus saline, one way ANOVA and Dunnett aspost-hoc test.

FIG. 4 Semi-quantitative evaluation of Fluoro-Jade staining −=nopositivity, +=low positivity, ++=intermediate positivity, +++=highpositivity.

FIG. 5 Representative images of neurodegeneration by Fluoro-Jadestaining in the striatum of ischemic mice receiving saline or 15 U/mouseof rabbit rhC1-INH at different time points from the onset of ischemia.Bar: 100 μm.

FIG. 6 Representative images of neurodegeneration by Fluoro-Jadestaining in the dentate gyrus of ischemic mice receiving saline or 15U/mouse of rabbit rhC1-INH at different time points from the onset ofischemia. Bar: 100 μm.

FIG. 7 Representative images of neurodegeneration by Fluoro-Jadestaining in the cortex of ischemic mice receiving saline or 15 U/mouseof rabbit rhC1-INH at different time points from the onset of ischemia.Bar: 100 μm.

FIG. 8 Infarct volume assessed 48 h after ischemia in mice receivingsaline or 5, 10, 15 U/mouse of rabbit rhC1-INH 3 hours after the onsetof ischemia. Data are expressed as mean±SEM (n=6 mice per group).**P<0.01 versus saline, one way ANOVA and Dunnett as post-hoc test.

FIG. 9 Infarct volume assessed 48 h after ischemia in mice receivingsaline or 15 U/mouse of pdC1-INH or cow, or rabbit rhC1-INH three hoursafter the onset of ischemia. Data are expressed as mean±SEM (n=6 miceper group). *P<0.05, **P<0.01 versus saline, one way ANOVA and Dunnettas post-hoc test.

FIG. 10 General (upper panel 10a) and focal (lower panel 10b) deficitsassessed 48 h after ischemia in mice receiving saline or 15 U/mouse ofpdC1-INH or cow, or rabbit rhC1-INH three hours after the onset ofischemia. (n=6 mice per group). **P<0.01 versus saline, one way ANOVAand Kruskal-Wallis as post-hoc test.

FIG. 11 Overview of the different pathways of complement activation.

FIGS. 12, 13 Effect of rhc1INH and pdC1INH on activation of theclassical complement pathway. Increasing doses of rhC1INH or pdC1INH(x-axis) was added to two different samples of normal human serum(sample 1 upper panel. sample 2 lower panel). As a control, the bufferin which rhC1INH is dissolved (20 mM citrate, 0.19 M sucrose pH 6.8;0.22 μm filtered) was taken along in the same dilutions as rhC1INH.Readout was deposition of C5b-9, the normal serum control in the assaydefining 100% (y-axis). Data are mean and SD (n=3).

FIGS. 14, 15 Effect of rhc1INH and pdC1INH on activation of the MBLcomplement pathway. Increasing doses of rhC1INH or pdC1INH (x-axis) wasadded to two different samples of normal human serum (sample 1 upperpanel. sample 2 lower panel). As a control, the buffer in which rhC1INHis dissolved (20 mM citrate, 0.19 M sucrose pH 6.8; 0.22 μm filtered)was taken along in the same dilutions as rhC1INH. Readout was depositionof C5b-9, the normal serum control in the assay defining 100% (y-axis).Data are mean and SD (n=3).

FIG. 16 Effect of rhc1INH and pdC1INH on activation of both theclassical and MBL pathway of complement activation. Increasing doses ofrhC1INH or pdC1INH was added to two different samples of normal humanserum. As a control, the buffer in which rhC1INH is dissolved (20 mMcitrate, 0.19 M sucrose pH 6.8; 0.22 μm filtered) was taken along in thesame dilutions as rhC1INH. Readout was deposition of C5b-9 and thepercentage complement activation was calculated per measurement withthis formula: (Sample−NC)/(PC−NC)×100. PC is set at 100%. Results shownare the mean SD of 3 independent verdunning at each concentrationtested.

EXAMPLES Example 1

Previous experiments showed that a single dose of rhC1INH (15 U/mouse)administered at the beginning of the ischemic period, significantlyreduces ischemic volume, as assessed 48 hours after ischemia in ourmouse model of cerebral focal ischemia in a manner very similar toplasma derived C1INH. In this Example we have explored the time windowof efficacy for rhC1INH neuro-protective activity on the ischemic volumeand functional deficits. We have also studied the effect of rhC1INH onseven-days outcome by assessing the neurodegeneration and glialresponse.

1. Methods 1.1 Transient Focal Cerebral Ischemia

Ischemia was achieved by middle cerebral artery occlusion (MCAO) aspreviously described (De Simoni et al., 2003 and 2004, supra).Anesthesia was induced by 5% isoflurane in N₂O/O₂ (70/30%) mixture andmaintained by 1.5-2% isoflurane in the same mixture. To confirm theadequacy of the vascular occlusion in each animal, blood flow wasmeasured by laser doppler flowmetry (Transonic BLF-21) using a flexible0.5 mm fiberoptic probe (Transonic, Type M, 0.5 mm diameter) positionedon the brain surface and secured with impression material on the skullat the following coordinates: AP=−1 mm; L=−3.5 mm. Briefly, the rightcommon carotid artery was exposed and a siliconized filament (7-0), wasintroduced into the internal carotid artery through an incisionperformed on the common carotid artery and advanced to the anteriorcerebral artery so as to block its bifurcation into the anteriorcerebral artery and the MCA. The filament was advanced until a >70%reduction of blood flow, compared to preischemic baseline, was observed.After 30 min of ischemia, blood flow was restored by carefully removingthe nylon filament.

1.2 Drug Treatment

Mice received a single iv injections of rhC1INH at the dose of 15U/mouse in 150 μl or the same volume of saline at different time fromischemia:

-   -   at the beginning of ischemic period (rhC1INH -pre).    -   at the end of ischemic period (rhC1INH -post).    -   one hour after the beginning of the ischemic period (rhC1INH 1 h        -post).

rhC1INH used in this study was produced in transgenic rabbits thatexpress human C1INH in their mammary glands and purified from the milkobtained from these animals as described in WO 01/57079.

1.3 Evaluation of Neurological Deficits

Forty-eight hours after ischemia, each mouse was rated on twoneurological function scales unique to the mouse, by a trainedinvestigator blinded to the experimental conditions. For generaldeficits mice were scored from 0 to 28 in each of the followingcategories: hair, ears, eyes, posture, spontaneous activity, epilepticbehavior. For focal deficits mice were scored from 0 to 28 in each ofthe following categories: body symmetry, gait, climbing, circlingbehavior, front limb symmetry, compulsory circling, sensory response.Data are expressed as median and 25^(th) to 75^(th) percentiles.

1.4 Quantification of Infarct Size

Forty-eight hours after ischemia, mice were deeply anesthetized withEquitensin (120 jil/mice, ip) and transcardially perfused with 30 ml ofPBS 0.1 mol/l, pH 7.4, followed by 60 ml of chilled paraformaldheyde(4%) in PBS. After carefully removing the brains from the skull, theywere transferred to 30% sucrose in PBS at 4° C. overnight forcryoprotection. The brains were then rapidly frozen by immersion inisopentane at −45° C. for 3 min before being sealed into vials andstored at −70° C. until use. For lesion size determination, 20 μmcoronal brain sections were cut serially at 240 μm intervals and stainedwith neutral red (Neutral Red Gun Certistain, BDH, England). On eachslice, infarcted areas were assessed blindly and delineated by therelative paleness of histological staining. The infarcted area wasdetermined by subtracting the area of the healthy tissue in theipsilateral hemisphere from the area of the contralateral hemisphere oneach section. Infarct volumes were calculated by the integration ofinfarcted areas on each brain slice as quantified with computer-assistedimage analyzer and calculated by Analytical Image System.

1.5 Open Field Test

Seven days after ischemia mouse behavior was evaluated by the open fieldtest. This test may be useful to dectect anxiety and exploratorybehavior, and locomor activity in long-term ischemic mice. The openfiled consisted of a plastic box (41×41×41 cm) containing 4 differentobjects. The area of the open field was divided into a 28×28 cm centralzone and the surrounding border zone. Mice were individually placed intothe centre of the open field and their behavior was observed for 5minutes by an investigator blinded to the experimental conditions. Thenumber of inside crossings (mainly related to anxiety behavior), outsidecrossings (mainly related to motor activity), rears (mainly related toexploratory behavior) and contacts with objects (mainly related tosensory/motor activity) was counted.

1.6 Neuronal Count

Seven days after ischemia, mice were transcardially perfused aspreviously described. For neuronal count determination, 20 μm coronalbrain sections were cut serially at 640 μm intervals and stained withcresyl violet (Cresyl Violet acetate, Sigma, St. Louis, Mo.). Three 20μm sections from ipsi- and controlateral hemispheres were selected forneuronal count. The first section was at stereotaxic coordinatesanteroposterior +0.86 from bregma. The amount of neuronal loss wascalculated by pooling the number of viable neurons in the three sectionsof both hemispheres and expressed as percentage of controlateralhemisphere. An Olympus BX61 microscope, interfaced with Soft ImagingSystem Colorview video camera and AnalySIS software was used. Thequantitative analysis was performed at 40× magnification by aninvestigator blinded to the treatment.

1.7 Immunohistochemistry for Astrocytes and Microglia

Seven days after ischemia twenty-μm-thick coronal sections fromtranscardially perfused ischemic mice were prepared and used forassessment of astrocytes and microglia/macrophages immunostainingBriefly, the sections were rinsed for 30 minutes in 0.4% Triton X-100 in0.1 mol/L PBS followed by 15 minutes in 0.1% Triton X-100 and 3% normalgoat serum (NGS) in PBS. The sections were then incubated overnight withantibody for astrocytes and microglia (anti-GFAP 1:1500, Chemicon;anti-CD11b 1:250, kindly gift by Dr. A. Doni, Mario Negri Institute).The next day, the sections were washed in PBS and incubated withbiotinylated secondary antibody for 1 h, washed and incubated withavidin-biotin-peroxidase. After reacting with 3′-3-diaminobenzidinetetrahydrochloride the sections were washed, dried, dehydratate throughgraded alcohols, fixed in xylene and coverslipped using DPX mountantbefore light microscopy analysis.

2. Results 2.1 Time-Window of Efficacy 2.1.1 Evaluation of NeurologicalDeficits

Neurological deficits were evaluated in ischemic mice receiving rhC1INHor saline 48 h after ischemia. A slight, although not significant,decrease in every group of rhC1INH-treated mice was observed compared tosaline-treated ischemic mice (rhC1INH -pre: 9 and 12; rhC1INH-post: 7and 11; rhC1INH 1 h -post: 9 and 13, saline: 10 and 12.5, median ofgeneral and focal deficits, respectively) (data not shown).

2.1.2 Assessment of Infarct Size

Forty-eight hours after ischemia rhC1INH-treated mice showed a markedreduction of the ischemic volume, at 15 U/mouse -pre, -post and 1 h-post doses (13.67±2.59 mm³, 9.06±0.77 mm³ and 8.24±1.00 mm³,respectively), compared to saline-treated mice (41.51±7.01 mm³) (FIG. 1,data are expressed as mean±SEM).

2.2 Seven-Days Outcome 2.2.1 Open Field Test

Ischemia induced a significant reduction in the number of rears comparedto naïve animals while in the rhC1INH-treated group this parameter wasnot different from non-ischemic mice. The other parameters evaluated didnot show any difference among the three groups.

2.2.2 Neuronal Count

To evaluate if the protective effect of rhC1INH is long lasting, weassessed the neuronal loss 7 days after induction of ischemia andtreatment with the drug. The results show that rhC1INH protective effectis still present at this time: 14%±2.18% versus 4%±1.24% mean of saline-and rhC1INH -treated mice, respectively (data not shown).

2.2.3 Immunohistochemistry for Microglia/Macrophages and Astrocytes

Seven days after ischemia a large amount of activated microglia andinfiltrated macrophages were observed in the lesioned hippocampus andstriatum of ischemic mice receiving saline (data not shown). Fifteenunits of rhC1INH-pre were able to counteract this activation andinfiltration in both the areas considered (data not shown). Theipsilateral hippocampus of saline-treated ischemic mice showed a slightastrocytosis which was not different from that observed inrhC1INH-ischemic mice (data not shown). Other brain areas did not showany relevant astrocytic activation in either groups.

3. Conclusions

The present data show that rhC1INH at the dose of 15 U/mouse issimilarly effective in reducing the ischemic volume when given at thebeginning (-pre) or at the end of ischemic period (-post, i.e. atreperfusion). More importantly the inhibitor is able to exert itsneuroprotective effects also when injected 1 hour after the onset ofischemia (1 h -post). Furthermore, the protective action of rhC1INH isstill present 7 days after ischemia. These results are in sharp contrastwith plasma derived hC1INH which when injected 1 hour after ischemia hasnearly completely lost the ability to exert neuroprotective effects (seeFIG. 2).

The main results of this study are the following:

1. the half life of rhC1INH in mouse plasma is about 3 hours (at a doseof 15 U/mouse). The good correlation between antigen and functionalactivity indicates that the recombinant protein circulates in plasma inits active form only; it is possible that tissue distributioncontributed to the reduction of plasma levels.

2. rhC1INH, at the dose of 15 U/mouse -pre is very effective in reducingthe ischemic volume (reduction of 69%).

3. rhC1INH at the dose of 15 U/mouse is able to clearly reduce thenumber of degenerating neurons in the hippocampus as assessed byFluoro-Jade staining thus indicating that the reduction in ischemicvolume is due to sparing of neurons.

4. rhC1INH is similarly effective in reducing ischemic volume when givenat the beginning (-pre), at the end of the ischemic period (-post, i.e.at reperfusion) or 1 hour after the onset of ischemia (1 h -post, i.e.30 min from beginning of reperfusion). Thus rhC1INH has a widertime-window of efficacy than pdC1INH (that is no more effective whengiven lh after ischemia).

5. the neuroprotective effect of rhC1INH-pre dose is long-lasting, asshowed by neuronal counting performed 7 days after the beginning ofischemia.

6. rhC1INH, induced a slight improvement of general and focal deficitsassessed 48 hours after ischemia. This finding is similar to whatobserved with pdC1INH. In order to evaluate the effect of rhC1INH onlong-term behavioral outcome, we analyzed mouse behavior by open fieldtest. Seven days after ischemia the rearing behavior shows asignificantly lower score in the ischemic compared to naïve mice. Thisdecrease is not present in rhC1INH treated mice whose score is notdifferent from control mice.

7. rhC1INH is able to counteract the activation/recruitment ofmicroglia/macrophages in ischemic mice brain as assessed both at early(48 h) and at late (7 days) time points. These cells are an index of theinflammatory response of the brain tissue.

8. The strong astrocytic response elicited by ischemia at 48 h isdampened by rhC1INH. The astrocytic activation is markedly decreased at7 days in both experimental groups and no difference between saline- andrhC1INH-treated mice could be observed.

Example 2 Study on the Neuroprotective Action of rhC1-INH in MouseModels of Focal Cerebral Ischemia

We have previously demonstrated that 15 U of rhC1-INH have a markedneuroprotective action in a model of murine cerebralischemia/reperfusion also when administrated 1 hour after the onset ofischemia/reperfusion, at variance with pdC1-ENH that, at this time ofpost-treatment, is no longer effective. This neuroprotection islong-lasting, in fact seven days after ischemia and treatment, ischemicbrains of mice treated with rhC1-INH still show a decreased infarctsize. In the following experiments we have determined the time window ofefficacy (beyond 1 hour post) and the dose-response of rhC1-INHneuroprotective activity on the ischemic volume. In addition we haveperformed a direct comparison among pdC1-INH, rabbit and cow rhC1-INH(at the most effective dose and time-points for rabbit rhC1-INH) usingthe same protocol.

Methods Animals

Procedures involving animals and their care was conducted in conformitywith institutional guidelines that are in compliance with national (D.L.n.116, G.U. suppl. 40, 18 Feb. 1992) and international laws and policies(EEC Council Directive 86/609, OJ L 358,1; Dec. 12, 1987; NIH Guide forthe Care and Use of Laboratory Animals, U.S. National Research Council1996). Male C57B1/6 mice (26-28 g, Charles River, Calco, Italy) werehoused 5 per cage and kept at constant temperature (21±1° C.) andrelative humidity (60%) with regular light/dark schedule (7 am-7 pm).Food (Altromin pellets for mice) and water available ad libitum.

Transient Focal Cerebral Ischemia

Ischemia was achieved by middle cerebral artery occlusion (MCAO) aspreviously described^(1,3). Anesthesia was induced by 5% isoflurane inNa0/0a (70/30%) mixture and maintained by 1.5-2% isoflurane in the samemixture. To confirm the adequacy of the vascular occlusion in eachanimal, blood flow was measured by laser doppler flowmetry (TransonicBLF-21) using a flexible 0.5 mm fiberoptic probe (Transonic, Type M, 0.5mm diameter) positioned on the brain surface and secured with impressionmaterial on the skull at the following coordinates: AP=−1 mm; L=−3.5 mm.Briefly, the right common carotid artery was exposed and a siliconizedfilament (7-0) was introduced into the internal carotid artery throughan incision performed on the common carotid artery and advanced to theanterior cerebral artery so as to block its bifurcation into theanterior cerebral artery and the MCA. The filament was advanced untila >70% reduction of blood flow,

compared to preischemic baseline, was observed. After 30 min ofischemia, blood flow was restored by carefully removing the nylonfilament.

Drug Treatment

Mice received a single iv injections of C1-INH (rabbit rhC1-INH, cowrhC1-INH or pdC1-INH) at different doses at different times fromischemia. Control mice received the same volume of saline.

Evaluation of Neurological Deficits.

Forty-eight hours after ischemia, each mouse was rated on twoneurological function scales unique to the mouse, by a trainedinvestigator blinded to the experimental conditions. For generaldeficits mice were scored from 0 to 28 in each of the followingcategories: hair, ears, eyes, posture, spontaneous activity, epilepticbehavior. For focal deficits mice were scored from 0 to 28 in each ofthe following categories: body symmetry, gait, climbing, circlingbehavior, front limb symmetry, compulsory circling, sensory response.Data are expressed as median and percentiles.

Quantification of Infarct Size

Forty-eight hours after ischemia, mice were deeply anesthetized withEquitensin (120 jil/mice, ip) and transcardially perfused with 30 ml ofPBS 0.1 mol/l, pH 7.4, followed by 60 ml of chilled paraformaldheyde(4%) in PBS. After carefully removing the brains from the skull, theywere transferred to 30% sucrose in PBS at 4° C. overnight forcryoprotection. The brains were then rapidly frozen by immersion inisopentane at −45° C. for 3 min before being sealed into vials andstored at −70° C. until use. For lesion size determination, 20 fjmcoronal brain sections were cut serially at 240 (j,m intervals andstained with neutral red (Neutral Red Gurr Certistain, BDH, England). Oneach slice, infarcted areas were assessed blindly and delineated by therelative paleness of histological staining. The infarcted area wasdetermined by subtracting the area of the healthy tissue in theipsilateral hemisphere from the area of the contralateral hemisphere oneach section. Infarct volumes were calculated by the integration ofinfarcted areas on each brain slice as quantified with computer-assistedimage analyzer and calculated by Analytical Image System.

Assessment of Neurodegeneration

The presence of neurodegeneration was evaluated on 20 jam thick sectionsby staining with Fluoro-Jade⁴, a marker for neuronal degeneration.Briefly, sections were dried and rehydrated in ethanol (100%-75%) anddistilled water. Then, they were incubated in 0.06% potassiumpermanganate for 15 minutes, washed in distilled water and transferredto 0.001% Fluoro-Jade staining solution for 30 minutes. After staining,the sections were rinsed in distilled water, dried, immerse in xyleneand coverslipped using DPX mountant (BDH, Poole, UK) before fluorescentmicroscopy analysis.

Results Time-Window Of Efficacy in Transient Ischemia

In order to evaluate the time-window of efficacy, 15 U of rabbitrhC1-INH or saline were given at 3, 6, 9, 18 and 24 hours from thebeginning of ischemia. Forty-eight hours later, ischemic mice treatedwith rabbit rhC1-INH 3 and 6 hours after the onset of ischemia showed amarked decrease of ischemic volume (11.71±0.63 mm³ and 20.38±2.37 mm³,respectively) compared to saline-treated ischemic mice (44.43±5.94 mm³).Also when administrated 9 and 18 hours after ischemia, rabbit rhC1-INHwas still effective, although to a minor extent (23.63±4.11 mm³ and27.13±2.58 mm³ respectively). Twenty-four hours after ischemia theinhibitor lost its beneficial action (41.92±2.76 mm³). (FIG. 3). Insaline-treated mice, Fluoro-Jade staining showed that, neurodegenerationwas present in striatum cortex and hippocampus. When administrated atearly time points, rhC1-INH was able to counteract the neurodegenerationin hippocampus (up to 3 hours) and in cortex (up to 9 hours). When micewere treated with this inhibitor 6 and 9 hours after ischemia, somedegenerating neurons were observed in hippocampus. At later time pointsof treatment (18 and 24 hours), when the ischemic volume was larger,Fluoro-Jade staining showed the presence of neurodegenerating neurons incortex. At all time points considered, striatum showed an extensiveneurodegeneration, both in saline- and rhC1-INH-treated animals (FIG. 5,6, 7). A semi-quantitative evaluation of Fluoro-Jade staining for eachanimal was performed by an investigator blinded to the experimentalconditions (FIG. 4).

Dose-Response in Transient Ischemia

Since the dose of C1-INH used in humans for hereditary angioedema islower than the one we used in mice for stroke treatment, lower doseswere used in our ischemic model. Based on the results of the previousexperiment we chose 3 h post treatment for dose-response experiment.Different doses of rabbit rhC1-INH (5 and 10 units) were given 3 hoursafter the onset of ischemia and reperfusion. The dose of 10 U/mouse wasstill effective in reducing the ischemic volume (22.10±3.65 mm³), while5 U of rabbit rhC1-INH did not modify the extent of the brain damage(47.39±4.08 mm³). These data show that rabbit rhC1-INH is able to modifythe ischemic lesion in a dose-dependent manner (FIG. 8).

In mice treated with 10 U of rhC1-INH some neurodegenerating neurons, asevidenced by Fluoro-Jade staining, were observed in striatum but not inhippocampus and cortex, while 5 U-treated ischemic mice displayed alarge neurodegeneration in striatum cortex (not shown).

General and focal neurological deficits did not show any significativevariations either in time-window of efficacy or in dose-responseexperiment (not shown).

COMPARISON BETWEEN THE EFFECT OF pdC1-INH and rhC1-INH (from rabbits andcows) Our previous data on pdC1-INH were obtained with a different modelof transient cerebral ischemia. In order to directly compare pdC1-INH,cow rhC1-INH and rabbit rhC1-INH, these compounds were given to mice inwhich ischemia was induced with the same experimental protocol(silicone-coated filament). The inhibitors were administrated at thedose of 15 U/mouse 3 hours after the onset of ischemia.

As expected, pdC1-INH was not able to exert a neuroprotective action atthis time point (47.39±4.08 mm³). At variance, cow rhC1-INH-treatedischemic mice showed a significantly reduced ischemic volume compared tosaline-treated mice, even though to a lower extent than rabbitrhC1-INH-treated mice (FIG. 9). Surprisingly both general and focaldeficits were significantly improved by cow rhC1-INH (FIG. 10).

Fluoro-Jade staining showed a large neurodegeneration in the brain ofischemic mice treated with pdC1-INH in all the considered areas (cortex,striatum and hippocampus). The staining of the brain of cowrhC1-INH-treated mice showed a variable grade of neurodegeneration incortex and hippocampus since in 3 out of 6 mice a markedneurodegeneration was observed in both these areas, while in the other 3mice the neurodegeneration was present in a very little amount. Thestriatum displayed an extensive Fluoro-Jade staining in 6 out of 6 mice.

Comments

The most relevant data of this work is the time-window of efficacy ofrabbit rhC1-INH. The dose of 15 U/mouse of rabbit rhC1-INH was able tosignificantly reduce the ischemic volume up to 18 hours after the onsetof ischemia at variance with pdC1-INH that 3 hours after ischemia hasalready lost its neuroprotective effect. This surprising feature makesrhC1-INH a possible candidate for stroke therapy in humans. Thedifferent efficacy of pd and rhC1-INHs, could be due to the differentglycosylation of the two molecules resulting in turn to a higheraffinity for a mannose binding protein (MBP) of rhC1-INH as compared tothe plasma derived one. Binding MBP, rhC1-MH causes the inhibition ofthe complement lectin pathway, involved in the pathogenesis of thedamage in heart, kidney and gastrointestinal ischemia/reperfusion^(7,9).The role of this poorly characterized pathway is still unknown in brainischemia and further experiments are required in order to clarify themechanism of rhC1-INH neuroprotection.

The superior neuroprotective effect of rhC1INH over pdC1INH in thetime-window after the onset of ischemia may further be explained by amore efficient targeting of the recombinant molecule to the site oftissue damage either through binding to cell-surface antigens and/or amore efficient tissue penetration. More research needs to be done tofully elucidate the exact molecular mechanism underlying the observationdescribed in this invention.

Fluoro-Jade staining gives indirect evidence of how the lesion evolvesin time. The early treatment with rhC1-INH provides a complete rescue ofthe ischemic penumbra (hippocampus and cortex). The later the treatmentis administrated, the more neurons in the penumbra degenerate. Thesefindings confirm that rhC1-INH exerts its neuroprotective action onischemic penumbra. Rabbit rhC1-INH is able to reduce ischemic volume ina dose dependent-manner. The most effective dose of rhC1-INH (15U/mouse, corresponding about to 600 U/kg), used for time-window ofefficacy experiment, is much higher than the one used in humans forhereditary angioedema (about 25-100 U/kg). In order to verify if a lowerdose was still effective in reducing neurodegeneration and ischemicinfarct, a dose-response experiment was performed. The results showedthat 400 U/kg (10 U/mouse) of rhC1-INH were still able to significantlycounteract the ischemic insult, although to a lower extent. A dose 8fold higher than the one used for HAE (5 U/mouse, 200 U/kg) was noteffective. These findings are in line with evidence showing that largedoses of C1-INH are required for therapeutic application in variousinflammation settings⁵. In particular such doses are necessary to reachan important inhibitory effect on endothelial adhesion molecules⁶, amechanism involved in the pathogenesis of ischemia/reperfusion braindamage. Lastly, cow rhC1-INH provided neuroprotection, when given 3hours after ischemia at the dose of 15 U/mouse, although less markedlythan rabbit rhC1-INH. The inhibitor from cow was also able to improveneurological deficits compared to saline-treated mice. These findingsindicate that this molecule is able to ameliorate the general conditionsof ischemic mice.

Example 3 Comparison of the Ability of rhC1INH and Plasma Derived C1INHto Inhibit Activation of the Classical and MBL Pathways Materials andMethods

The effect of rhC1INH and pdC1INH (Cetor, Sanquin, Amsterdam, TheNetherlands) on the function of the classical and lectin pathway wasexamined in the Wieslab TM complement system Screen (Euro-Diagnostica,Malmø, Sweeden) using two different sources of serum. One serum sourceis included in the kit, where it is used as a positive control(hereafter referred to as serum sample 1). The other serum sample wasobtained from a commercially available pool of human serum (pool of 25different donors; Kordia, Leiden, The Netherlands), hereafter referredto as serum sample 2. Both serum samples were incubated in independenttriplo's with 0, 15, 30 and 75 μmol rhC1INH or pdC1INH for 30 min atroom temperature. Therefore, stock solutions of pdC1INH and rhC1INH werediluted in water to appropriate concentrations. Volumes correspondingwith 15, 30 and 75 μmol rhC1INH or pdC1INH were taken and adjusted to 15μl with water. The buffer in which rhC1INH is dissolved (20 mM citrate,0.19 M sucrose pH 6.8; 0.22 μm filtered) was taken along in the samedilutions as rhC1INH to control for interference with the WieslabComplement System. The positive controls (PC) and negative controls (NC)from both the classical and MBL pathway (provided with the kit), andboth serum samples were diluted 1/101 in Diluent CP for the classicalpathway and Diluent MP for the MBL pathway according to manufacturer'sinstructions. Of these diluted sera, 127.5 μl was supplemented with 22.5μl water, pdC1INH, rhC1INH or buffer and incubated for 30 minutes at RT.Next, 100 μl/well of PC, NC, Diluent CP or MP (blanks) and samples werepipetted on the appropriate plate and incubated for 1 hour at 37° C.After incubation the wells were washed 3 times with 300 μl/well washingsolution and subsequently incubated for 30 minutes at room temperaturewith 100 μl/well of conjugate. After another wash, wells were incubatedwith 100 μl/well of substrate and again incubated for 30 minutes at roomtemperature.

The reaction was stopped by the addition of 100 μl/well 5 mM EDTA andthe absorbance was read at 405 nm.

For the calculation of the results, the absorbance of the blanks(Diluent CP or MP) was subtracted from the PC, NC and samples. Thepercentage complement activation was calculated per measurement withthis formula: (Sample−NC)/(PC−NC)×100. This means that the PC is alwaysset at 100%. For each condition the mean, standard deviation and CV %was calculated.

Results

Effect of rhC1INH and pd-C1INH on the Classical Pathway as Examined byWielisa.

The inhibitory properties of both rhC1INH and pdC1INH on classicalpathway activation were analyzed in two different serum samples. Asshown in FIGS. 12, 13 and 16, both rhC1INH and pdC1INH dose-dependentlyreduced the classical pathway mediated C5b-9 deposition in both serasamples. Whereas rhC1INH—at a concentration of 75 μM—seems to inhibitthe classical pathway activation in serum 1 slightly stronger thanpdC1INH, such an effect was not seen in serum sample 2. At all otherconcentrations tested no differences in inhibitory properties wereobserved between rhC1INH and pdC1INH. Therefore, it was concluded thatboth rhC1INH and pdC1INH are equally effective in inhibiting classicalpathway activation in human sera.

Effect of rhC1INH and pdC1INH on the MBL Pathway as Examined by Wielisa.

In the same set of experiments, also the inhibitory properties of bothrhC1INH and pdC1INH on the MBL pathway activation were analyzed. Asshown in FIGS. 14, 15 and 16, both rhC1INH and pdC1INH alsodose-dependently reduced activation of the MBL pathway. However, incontrast to the classical pathway where no differences were seen,rhC1INH appeared to be a more potent inhibitor of the MBL pathway whencompared to pdC1INH. At all 3 concentrations tested and in both serumsamples, the rhC1INH-mediated inhibition of the MBL pathway is ˜20%higher as compared to pdC1INH. Therefore, it was concluded that rhC1INHis a more effective inhibitor of the MBL pathway than pdC1INH.

CONCLUSION

The results show that both rhC1INH and pdC1INH are equally effective ininhibiting the classical pathway, but rhC 1INH is a more potentinhibitor of the MBL pathway. At all concentrations tested, rhC1INHmediated MBL pathway inhibition was ˜20% stronger as compared topdC1INH.

REFERENCES

-   1. De Simoni, M. G. et al. Neuroprotection by complement (C1)    inhibitor in mouse transient brain ischemia. J Cereb Blood Flow Met    23, 232-239 (2003).-   2. De Simoni, M. G. et al. The powerful neuroprotective action of    C1-inhibitor on brain ischemia-reperfusion injury does not require    C1q. Am J Pathol 164, 1857-63 (2004).-   3. Storini, C. et al. C1 inhibitor protects against brain    ischemia-reperfusion injury via inhibition of cell recruitment and    inflammation. Neurobiol Disease 19, 10-17 (2005).-   4. Schmued, L. C. & Hopkins, K. J. Fluoro-Jade B: a high affinity    fluorescent marker for the localization of neuronal degeneration.    Brain Res 874, 123-30. (2000).-   5. Caliezi, C. et al. C1 esterase inhibitor: an anti-inflammatory    agent and its potential use in the treatment of diseases other than    hereditary angioedema. Pharmacol Rev 52, 91-112 (2000).-   6. Cai, S. et al. A direct role for C1 inhibitor in regulation of    leukocyte adhesion. J Immunol 174, 6462-6 (2005).-   7. Walsh, M. C. et al Mannose-binding lectin is a regulator of    inflammation that accompanies myocardial ischemia and reperfusion    injury. J Immunol 175, 541-6 (2005).-   8. Moller-Kristensen, M. et al. Mannan-binding lectin recognizes    structures on ischaemic reperfused mouse kidneys and is implicated    in tissue injury. Scand J Immunol 61, 426-34 (2005).-   9. Hart, M. L. et al. Gastrointestinal ischemia-reperfusion injury    is lectin complement pathway dependent without involving C1q. J    Immunol 174, 6373-80 (2005).

1. A method of preventing, reducing or treating at least one of ischemiaand reperfusion injury in a patient comprising administering to thepatient a C1 inhibitor having a plasma half life of less than 6 hours,whereby the C1 inhibitor is administered after the onset of ischemiaand/or reperfusion.
 2. The method according to claim 1, wherein the C1inhibitor has a reduced level of terminal sialic acid residues ascompared to plasma derived human C1 inhibitor.
 3. The method accordingto claim 1, wherein the C1 inhibitor comprises a glycan that has aterminal residue selected from galactose, N-acetylgalactosamine,N-acetylglucosamine, mannose and fucose.
 4. (canceled)
 5. The methodaccording to claim 1, wherein the C1 inhibitor is obtained from agenetically engineered cell or organism.
 6. The methods according toclaim 5, wherein the C1 inhibitor is obtained from a transgenicnon-human animal.
 7. The method according to claim 6, wherein the C1inhibitor is obtained from the milk of said transgenic non-human animal.8. The method according to claim 6, wherein the transgenic non-humananimal is a bovine or an animal of the order Lagomorpha, preferably arabbit.
 9. The method according to claim 1, wherein the C1 inhibitor isused in an amount in the range of 50-2000 units per kg body weight. 10.The method according to claim 1, wherein the C1 inhibitor isadministered at least one hour after the onset of ischemia and/orreperfusion, preferably at least 3 hours, more preferably at least 6hours, more preferably at least 9 hours, even more preferably at least18 hours.
 11. The method according to claim 1, wherein the C1 inhibitoris administered in combination with a thrombolytic agent or aftertreatment with such agent.
 12. The method according to claim 1, whereinthe patient has at least one unforeseen sudden or acute occurrence ofischemia and/or reperfusion injury.
 13. The method according to claim12, wherein the C1 inhibitor administration prevents, reduces or treatsat least one of ischemia and reperfusion injury after stroke orperinatal stroke in the patient.
 14. The method according to claim 1,wherein the patient has at least one foreseen occurrence of ischemiaand/or reperfusion injury, preferably after organ transplantation.
 15. Amethod for the prevention, reduction or treatment of at least one ofischemia and reperfusion injury in a patient, comprising administering aC1 inhibitor having a plasma half-life of less than 6 hours to thepatient wherein the inhibitor is administered before or during at leastone foreseen occurrence of ischemia and/or reperfusion injury in thepatient.
 16. The method according to claim 15, wherein the C1 inhibitoris administered at the most 3 hours before at least one foreseenoccurrence of ischemia and/or reperfusion injury, preferably at the most2 hours, more preferably at the most 1 hour, and most preferably at themost 30 minutes and/or wherein the C1 inhibitor of the invention iscontinuously administered to a subject in the need thereof and/or incase of an organ transplantation to the organ to be transplanted. 17.The method according to claim 15, wherein the foreseen occurrence ofischemia and reperfusion injury is organ transplantation.
 18. The methodaccording to claim 17, wherein the pharmaceutical composition is for theprevention, reduction or treatment of at least one of ischemia and/orreperfusion injury known to arise via the lectin pathway, preferablymyocardial, renal, gastrointestinal ischemia and reperfusion injury orstroke.
 19. The method according to claim 13, wherein the C1 inhibitorexerts a neuroprotective effect preferably in the hippocampus and/orcortex.
 20. The method according to claim 13, wherein the C1 inhibitorexerts a reduction of a lesion induced by the ischemia and/orreperfusion.